WO2006001848A2

WO2006001848A2 - Quantum dots as high-sensitivity optical sensors and biocompatible imaging probes, compositions thereof, and related methods

Info

Publication number: WO2006001848A2
Application number: PCT/US2005/004310
Authority: WO
Inventors: Christoph A. Naumann; Bruce A. Young; Eric C. Long
Original assignee: Advanced Research And Technology Institute, Inc.
Priority date: 2004-02-12
Filing date: 2005-02-11
Publication date: 2006-01-05
Also published as: WO2006001848A3

Abstract

The invention provides a semiconductor nanoparticle with improved stability and biocompatibility properties. Accordingly, the present invention provides a nanoparticle that is capped with two separate protective layers: an inner inert layer and an outer inert layer and related in vitro and in vivo methods of use.

Description

QUANTUM DOTS AS HIGH-SENSITIVITY OPTICAL SENSORS AND BIOCOMPATIBLE IMAGING PROBES, COMPOSITIONS THEREOF, AND RELATED METHODS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/543,965, filed February 12, 2004, which is incorporated herein by reference.

FIELD OF THE INVENTION [0002] This invention pertains to a quantum dot comprising (a) an inner inert layer; and (b) an outer inert layer, in which the outer inert layer is optionally conjugated to a biological agent.

BACKGROUND OF THE INVENTION [0003] Quantum dots are defined as small particles whose linear dimension in all three directions is less than the de Broglie wavelength of the exciton (electron/hole pair). Such particles have a greatly modified electronic structure from the corresponding bulk semiconductor material and, in particular, the density of states becomes more like that for molecules. The applications for quantum dots are generally in the field of optoelectronics, such as light switches and light emitters. General reviews of quantum dots and their properties are known in the literature (see, for example, Weller, Angewandte Chemie International Edition (English) 1993, 32, 41-53: "Semiconductor q-particles: chemistry in the transition region between solid state and molecules"). [0004] Recent advances in bioanalytical sciences and bioengineering have led to the development of devices that use quantum dots, including DNA chips, miniaturized biosensors and microfluidic devices. In addition, applications benefiting from fluorescent labeling with conventional organic dyes or quantum dots include medical and non-medical fluorescence microscopy, histology, flow cytometry, fundamental cellular and molecular biology protocols, fluorescence in situ hybridization, DNA sequencing, immuno assays, binding assays and separation. These enabling technologies have substantially impacted many areas in biomedical research, such as gene expression profiling, drug discovery, and clinical diagnostics. For example, a conjugate, in which a quantum dot is linked to a probe moiety that has an affinity for a biological target, can be used as sensors to detect the presence or amounts of a biological moiety; the structure, composition, and conformation of a biological moiety; the localization of a biological moiety in an environment; interactions of biological moieties; alterations in structures of biological compounds; and alterations in biological processes. [0005] In comparison to organic dyes (e.g., Rhodamine), which traditionally have been used to detect biological targets, quantum dots are 20 times as bright, approximately 100 times as photostable, and have emission spectra that are approximately one third the width. Over the past decade, much progress has been made in the synthesis and characterization of a wide variety of semiconductor quantum dots. Recent advances have led to large-scale preparation of relatively monodisperse quantum dots (Murray et al., /. Am. Chem. Soc, 115, 8706-15 (1993); Bowen Katari et al., J. Phys. Chem., 98, 4109-17 (1994); and Hines et al., J. Phys. Chem., 100, 468-71 (1996)). Other advances have led to the characterization of quantum dot lattice structures (Henglein, Chem. Rev., 89, 1861-73 (1989); and Weller et al., Chem. Int. Ed. Engl. 32, 41-53(1993)) and also to the fabrication of quantum-dot arrays (Murray et al, Science, 270, 1335-38 (1995); Andres et al., Science, 273, 1690-93 (1996); Heath et al., J. Phys. Chem., 100, 3144-49 (1996); Collier et al., Science, 277, 1978-81 (1997); Mirkin et al., Nature, 382, 607-09 (1996); and Alivisatos et al., Nature, 382, 609-11 (1996)) and light-emitting diodes (Colvin et al., Nature, 370, 354-57 (1994); and Dabbousi et al., Appl. Phys. Let., 66, 1316-18 (1995)). [0006] The sensitivity of sensors toward particular molecules is often limited by the fact that they lack specificity due to unspecific reactions between other molecules and the sensor surface. The sensitivity of the sensor is, therefore, strongly linked to its ability to suppress such unspecific reactions. This aspect is particularly challenging for complex mixtures of molecules, as commonly found in biological systems. Therefore, sensors are needed which promote specific binding reactions yet prevent unspecific binding reactions with great efficiency. The efficient suppression of unspecific adsorption with biomolecules also is of great relevance for the proper function of imaging probes for in vivo imaging applications. In this case, it is necessary to use probes which bind particular biomolecules with great specificity, thereby preventing probe aggregation and unspecific binding to other biological agents. [0007] The invention describes strategies for the proper design of metallic and semiconductor nanoparticles ("quantum dots") which prevent the unspecific adsorption of molecules or molecular assemblies in complex mixtures of molecules, such as those found under in vivo conditions, but promote specific binding to particular target molecules or molecular assemblies. A novel feature of the proposed design is that system stability will be achieved by designing proper surface coatings that are covalently coupled to the core of the nanoparticle. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION [0008] Though conjugation strategies to biological agents, such as proteins, have been developed, the application for imaging and sensing studies under in vivo conditions is still very limited because existing systems, which rely on the coating of one protective layer, lack sufficient stability and inertness to prevent nanoparticle aggregation and unspecific adsorption of biomolecules to those nanoparticles. A key concept of this invention is to improve stability and biocompatibility properties by capping the nanoparticle with two separate protective layers: an inner inert layer and an outer inert layer. This layer design addresses the fact that the adsorption of biomoleules on substrates is linked to the entropy and enthalpy properties of the substrate (McPearson P roc. Natl. Acad. ScL USA 2000, 10, 1073). [0009] Thus, the invention provides a quantum dot comprising (a) an inner inert layer; and (b) an outer inert layer, in which the outer inert layer is optionally conjugated to a biological agent. [0010] The present invention also provides a method of detecting the location of a target within a sample. The method comprises (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the sample or a section thereof, thereby detecting the location of the target within the sample. [0011] Also provided by the present invention is a method of monitoring a biological process in vitro. The method comprises (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, wherein the target functions in a biological process, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the sample or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vitro. [0012] The present invention provides a method of detecting the location of a target in vivo. The method comprises (i) administering to a host a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the host, (ii) allowing the biological agent to specifically bind to the target, (iii) imaging the host, a section thereof, or a cell thereof, thereby detecting the location of the target in vivo. [0013] The present invention provides a method of monitoring a biological process in vivo. The method comprises (i) administering to a host a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the host, wherein the target functions in a biological process, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the host, a section, or a cell thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vivo. [0014] The present invention also provides a method of detecting the location of more than one target within a sample. The method comprises (i) contacting a sample with two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots of either series is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the sample, (ii) allowing the biological agents to specifically bind to the targets, (iii) imaging the sample or a section thereof, thereby detecting the location of the more than one target within the sample. [0015] Further provided by the present invention is a method of monitoring a biological process in vitro. The method comprises (i) contacting a sample with two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots of either series is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the sample, wherein each of the targets functions in a biological process, (ii) allowing the biological agents to specifically bind to the targets, and (iii) imaging the sample or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vitro. [0016] A method of detecting the location of more than one target in vivo is provided by the present invention. The method comprises (i) administering to a host two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots of either series is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the host, (ii) allowing the biological agents to specifically bind to the targets, (iii) imaging the host, a section thereof, or a cell thereof, thereby detecting the location of the more than one target in vivo. [0017] The present invention also provides a method of monitoring a biological process in vivo. The method comprises (i) administering to a host two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots of either series is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the host, wherein each of the targets functions in a biological process, (ii) allowing the biological agents to specifically bind to the targets, and (iii) imaging the host, a sample thereof, or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS [0018] Figure 1 is a schematic illustration of a quantum dot ("core") comprising an inner inert layer, an outer inert layer, wherein the outer inert layer is covalently bound to a biological agent (indicated as R), such as a biomolecule. [0019] Figure 2 is a schematic illustration of the formation of an inner inert layer on a quantum dot. [0020] Figure 3 is a schematic illustration of the formation of an inner inert layer on a quantum dot using two different molecules. [0021] Figure 4 is a schematic illustration of the "grafting to" process of forming the inner inert layer. [0022] Figure 5 is a schematic illustration of replacing the trioctylphosphine oxide (TOPO) passivating layer with mercaptopropionic acid (MPA) or a mixture of MPA and mercaptoethanol (ME). [0023] Figure 6 is a schematic illustration of the "grafting to" process of forming the inner inert layer using X-Si-F₁. [0024] Figure 7 is a schematic illustration of the "grafting from" process of forming the inner inert layer. [0025] Figure 8 is a schematic illustration of the formation of an inner inert layer comprising two different moieties. [0026] Figure 9 is a schematic illustration of the formation of the outer inert layer with F₄-S₄-F₅.

DETAILED DESCRIPTION OF THE INVENTION [0027] The invention provides a quantum dot comprising (a) an inner inert layer; and (b) an outer inert layer, in which the outer inert layer is optionally conjugated to a biological agent (Figure 1). [0028] As will be appreciated by the ordinary skilled artisan, the term "quantum dot" ("QD") in the present invention is used to denote a semiconductor nanocrystal. Each QD typically comprises a core and a passivating layer comprised of different materials, although QDs comprising only one type of material are encompassed by the present invention. Generally, however, the fluorescence emission increases when a passivating layer is used. Regardless of whether a single material or a core/passivating layer structure is used, the entire QD preferably has a diameter ranging from about 0.5 nm to about 30 nm, and more preferably from about 1 nm to about 10 nm, and most preferably from about 2 nm to about 5 nm. [0029] The "core" is a nanoparticle-sized semiconductor. While any core of the II- VI semiconductors (e.g., ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, and mixtures thereof), III-V semiconductors (e.g., GaAs, InGaAs, InP, InAs, and mixtures thereof) or IV (e.g., Ge, Si) semiconductors can be used in the context of the present invention, the core must be such that, upon combination with a passivating layer, a luminescent quantum dot results. A II- VI semiconductor is a compound that contains at least one element from Group II and at least one element from Group VI of the periodic table, and so on. Preferably, the core is a IIB-VIB semiconductor, a IIIB-VB semiconductor or a IVB-IVB semiconductor that ranges in size from about 1 nm to about 10 nm. The core is more preferably a IIB-VIB semiconductor and ranges in size from about 2 nm to about 5 nm. Most preferably, the core is CdS or CdSe. [0030] The synthesis of QDs is well known in the art as disclosed, for example, by U.S. Patents Nos. 5,906,670, 5,888,885, 5,229,320, 5,482,890, International Patent Application No. PCT/US0321878, and Hines, M. A. /. Phys. Chem., 100, 468-471 (1996), Dabbousi, B. O. J. Phys. Chem. B, 101, 9463-9475 (1997), Peng, X., J. Am. Chem. Soc, 119, 7019-7029 (1997), which are incorporated herein by way of reference. [0031] The wavelength emitted by the QDs can be selected according to the physical properties of the QDs, such as the size of the nanocrystal. QDs are known to emit light from about 300 nm to about 1700 nm. The wavelength band of light emitted by the QD is determined by either the size of the core or the size of the core and passivating layer, depending on the materials comprising the core and passivating layer. The emission wavelength band can be tuned by varying the composition and the size of the QD and/or adding one or more passivating layers around the core in the form of concentric shells. [0032] The passivating layer comprises a material that differs from the semiconductor of the core and binds to the core, thereby forming a surface layer or shell on the core. The passivating layer must be such that, upon combination with a given semiconductor core, results in a luminescent quantum dot. In general, the passivating layer passivates the core by having a higher band gap than the core, so the excitation of the QD is confined to the core, thereby eliminating nonradiative pathways and preventing photochemical degradation. [0033] In one embodiment, the passivating layer is preferably a IIB-VIB semiconductor of high band gap. More preferably, the passivating layer is ZnS, CdS, CdSe, CdTe, GaAs, or AlGaAs. Most preferably, the passivating layer is ZnS. In particular, the passivating layer is preferably ZnS when the core is CdSe or CdS and the passivating layer is preferably CdS when the core is CdSe. Other examples of core/passivating layer combinations for QDs include CdS/HgS/CdS, InAs/GaAs, GaAs/ AlGaAs and CdSe/ZnS. [0034] In another embodiment, the passivating layer comprises an organic moiety such as, for example, a thiol-containing monomer, such as an alkylthiol (CH₃(CH₂)_nSH, where n = 1-20, inclusive) (e.g., 1-nonanethiol) or a substituted alkylthiol (e.g., 2-aminoethanethiol), an amino-containing monomer, or phosphine-containing monomer (e.g., trioctylphosphine (TOP) and trioctylphosphine oxide (TOPO)). [0035] Regardless of the type of passivating layer used, it is essential that the quantum dots are prepared such that reactive surface groups (e.g., Q and Q' as used herein), such as amino (-NH₂, or NHR, wherein R is an alkyl or aryl group), carboxy (-COOH), thiol (-SH), or succinimidyl ester are located on the outside of the passivating layer. These reactive groups allow for covalent coupling of spacer molecules to form the inner inert layer. [0036] Because of the nature of the outer inert layer, some molecules could diffuse to the underlying surface over time; therefore, the inner layer should exhibit inert surface properties. Preferably the inner inert layer is formed by adding spacer molecules such that one end of the spacer molecule is reactive to the terminal group of the quantum dot and the other end of the spacer molecule is such that it provides a layer with inert surface properties. In order to provide inert surface properties, functional groups preferably are electrically neutral, hydrophilic, and/or hydrogen bonding acceptors. Generically, the spacer molecules that form the inner inert layer are of the formula: Y-Si-Fi, wherein Y is reactive with a group found on the surface of the quantum dot (e.g., "Q"), Si is a spacer, and Fi is a functional group that provides inert surface properties (Figure 2). Preferably Y is any group that can react with the functional groups found on the surface of the quantum dot, such as, for example, amino, isothiocyano, haloacetyl (e.g., iodoacetyl), benzyl halide groups, alkyl halide groups, maleimido, aziridino, acryloyl, an acrylating agent, or a thiol-disulfide exchange reagent. [0037] Si is any suitable spacer, such as a hydrocarbon chain, a hydrophilic polymer, or polypeptide chain of well-defined amino acid sequence. A suitable hydrocarbon chain has the formula -(CH₂V, ^m which n is 1 to 30, inclusive. Preferably, n is 1 to 25, more preferably n is 1 to 15, more preferably n is 1 to 10, and most preferably n is 1 to 6. A suitable hydrophilic polymer includes those discussed herein, in particular with reference to the outer inert layer. A suitable polypeptide chain comprises any amino acid unit, especially the twenty naturally occurring amino acids (e.g., lysine, alanine, etc.). The polypeptide chain can be of any length, such as, comprising two or more amino acid units. Preferably, the polypeptide chain contains 2 to about 15 amino acid units, more preferably it contains 2 to about 10 amino acid units, more preferably it contains 2 to about 8 amino acid units, more preferably it contains 2 to about 6 amino acid units, and most preferably it contains 2 to about 4 amino acid units. [0038] In one embodiment, inner inert surface coatings can be designed which consist of a mixture of molecules comprising those with an end-functional group Fi (i.e., Y-Si-Fj) and those characterized by an end-functional group F₂ (i.e., Z-S₂-F₂) (Figure 3). Z, S₂, and F₂ have the same definitions as Y, Si, and Fi, respectively. Y and Z are the same or different, but preferably, Y and Z are the same reactive group for covalent coupling of the molecules to the nanoparticle surface. Si and S₂ are the same or different spacer groups to control the relative distance each functional group extends from the surface. F₂ represents a reactive group, which can be used to attach the second inert layer and/or molecules facilitating the specific binding of other molecules to the nanoparticle. For steric reasons, the spacer of the F₂-carrying molecule preferably is at least as long as the spacer of the Fi- carrying one. More preferably S₂ is longer than Si. In some preferred embodiments, more complex mixtures are desirable, such as those containing Y-Si-Fi, Y-S₂-F₂, Y-S₃-F₃, Y-S₄- F₄, etc. [0039] When using polymer spacers, two grafting approaches are possible, via "grafting to" of the end-functionalized polymer to the nanoparticle surface or via "grafting from" polymerization of a particular polymer from the surface. In the case of "grafting to" the nanoparticle surface adsorbs coiled polymer chains, which results in a relatively low polymer density of the adsorbed polymer layer (see Figure 4). Since high grafting density is preferable for the proper function of the inner inert layer, the "grafting to" approach preferably is limited to oligomers. More preferably, the spacers on the inner inert layer are comprised of very short polymer chains containing not more than about 10 monomer segments. [0040] For example, a QD with hydrophilic surface groups prepared by the "grafting to" process is as follows. A quantum dot was prepared with a TOPO passivating layer, which was replaced with a layer of mercaptopropionic acid (MPA) or a mixture of MPA and mercaptoethanol (ME) (see Figure 5). An inner inert layer was formed by attaching an additional layer comprising molecules that have one end group that is reactive towards the passivation layer (e.g., MPA or MP A/ME), a spacer group, and a hydrophilic surface group (i.e., X-Si-Fi) (see Figure 6). [0041] Another approach is to covalently attach peptides comprising well-controlled amino acid sequences to the nanoparticle via peptide bond formation through a peptide- derived amine or carboxylic acid functionality. Alternatively, peptides of defined sequence can be covalently attached directly to the nanoparticle surface via the inherent affinity of a cysteine sulfhydryl or conjugated through the formation of a disulfide crosslink to an existing sulfhydryl found on the surface of the nanoparticle. In yet another possibility, an amine functionalized nanoparticle surface can be employed as the starting residue in a solid phase peptide synthesis protocol in which the nanoparticle provides a starting point/surface for peptide syntheses of any sequence. The peptide sequence is as described herein. [0042] In the case of "grafting from," preferably an initiator molecule is bound to the surface of the stabilizing layer. Monomer is added to the initiator, and the polymer is grown to the desired length (see Figure 7). Typically, the resulting grafted polymers are in a stretched conformation (Le., a polymer brush), therefore, allowing for high polymer densities. In addition, this approach typically results in stretched polymer conformations for polymer chains of more than 10 monomers, which are otherwise in a polymer coil. The graft density is only limited by the initiator surface density. [0043] If necessary, one or more coupling reagents can be used to covalently attach the inner inert layer to the quantum dot surface. For example, l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (EDC) is a water-soluble derivative of carbodiimide that catalyzes the formation of amide bonds between carboxylic acids or phosphates and amines by activating carboxyl or phosphate to form an O-urea derivative. This derivative reacts readily with nucleophiles. The reagent can be used to make ether links from alcohol groups and ester links from acid and alcohols or phenols, and peptide bonds from acid and amines. Carbodiimide is often used in the synthesis of peptides as the water-soluble derivative EDC or as the organic soluble derivative, N.W-dicyclohexyl-carbodiimide (DCC). N-Hydroxysuccinimide (NHS) is often used to assist the carbodiimide coupling in the presence of EDC. The carbodiimide coupling in the presence of EDC (sometimes also in the presence of NHS) is usually performed in the solution of HEPES-buffer (N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid) at optimal pH = 7.2-7.5. [0044] For example, a quantum dot ("R") with carboxy surface groups (Le., "Q") could be covalently bound to a spacer molecule to form an inner inert layer.

EDC R-COOH + NH₂ (C₂H₄O)_nH > R-CONH (C₂H₄O)_nH + urea

[0045] Nanoparticles capped with an inner inert layer can be prepared as described above. The surface of the inner inert layer should contain some reactive surface groups (e.g., Fi, F₂, and/or F₃) such as carboxy or succinimidyl esters. Polymers are added which contain the complementary functional group for. covalent coupling of the polymer to the surface groups (e.g., Fi, F₂, and/or F₃). Surface functional groups of the stabilizing inner inert layer not involved in covalent coupling to the polymer layer preferably are electrically neutral, hydrophilic, and acceptors for hydrogen bonding. [0046] After the inner inert layer is formed, the outer inert layer is added using molecules of the general formula F₄-S₄-F₅. F₄ is a group that is reactive to Fi, F₂, and/or F₃. S₄ is a spacer and F₅ can conjugate to one or more biological agents. To prevent aggregation of nanoparticles under physiological conditions, preferably the outer inert layer comprises one or more hydrophilic polymers (S₄). More preferably, the outer layer comprises flexible hydrophilic polymer chains of sufficient molecular weight to form polymer coils. Typically, nanoparticle aggregation is prevented because flexible polymer chains act as entropic springs, thereby repelling each other. To avoid the adsorption of molecules at the surface, the adsorption process should be energetically unfavorable with respect to both enthalpy and entropy. Moreover, the polymer molecular weight of the outer inert layer preferably is high enough to allow for the formation of a highly entropic surface. The high entropy of the outer layer ensures that no entropy-driven adsorption events can occur. Suitable hydrophilic polymers (S₄) are linear, branched, and/or cross-linked and include hydroxyethyl cellulose, glycosaminoglycans, dextran, dextran sulfate, polyethyleneimine, polyacrylamide, polyester, polyvinyl alcohol (PVA), poly(N- vinylpyrrolidine) (PVP), poly(N-vinylamide), poly(N-vinyl saccharide), poly(aminoacrylate), poly(sodium acrylate), poly(sodium methacrylate), poly(sodium styrenesulfonate,) polyurethane, polyethylene glycol (PEG), polyethylene oxide (PEO), poly(2-hydroxyethyl methacrylate) (poly HEMA), poly(2-(hydroxyethoxy)ethyl methacrylate) (poly DEGMA), poly(2-(acetoxy)ethyl methacrylate (polyAEMA), mixtures thereof, and copolymers thereof. Preferably the hydrophilic polymer is PEG, PEO, or mixtures thereof. Especially preferred polymers are hydrophilic and do not carry a net charge, such as PEG. For applications requiring a high degree of water solubility, polymers with a negative net charge (e.g., dextrans) or polymers found in the extracellular matrix (e.g., glycosaminoglycans) can be used. Alternatively, polymer coatings formed on the basis of photoreactive groups could be used to form larger particles comprising multiple nanoparticles embedded into a polymer matrix. [0047] To allow for conjugation reactions to one or more biological agents, the polymer can also comprise one or more of the following functional groups (F₅): (1) reactive groups, such as NHS, for conjugation of biological agents; (2) functional groups which are sensitive to environmental conditions, such as pH, concentration of specific solutes, or changed entropy of the polymer due to the specific binding to biological interfaces; and/or (3) photoreactive groups, such as benzophenone. The term "sensitive" implies that as a result of one or more environmental stimuli, the polymer properties can change, such as the degree of swelling, entropy of the polymer (sol-gel transition), or net charge of the polymer. Polymers that are stimuli-responsive include hydrogels and thermoreversible gels. [0048] The quantum dots of the present invention or of the present inventive series of quantum dots can be conjugated to a biological agent. By "conjugated" as used herein means that the quantum dot is attached to a biological agent through any means, e.g., chemical bonds, electrostatic interactions, cross-linkers, and the like. As used herein the term "biological agent" refers to any molecule, entity, or part of either of the foregoing that is endogeneous to a whole organism and/or is biologically active within a whole organism. Suitable biological agents for conjugation to the present inventive quantum dots are known in the art and include, for instance, a biomolecule or a drug. Preferably, the biological agent is a biomolecule, wherein "biomolecule" refers to any molecule or part thereof that is naturally-occurring within or on the body of a whole organism. Preferred biomolecules for conjugation to the present inventive quantum dots include a protein, a peptide, a nucleic acid molecule, a combination thereof, and the like. Also preferred is that the biological agent is a drug, wherein "drug" as used herein refers to any chemical agent that is exogeneous to the body of a whole organism and typically is synthesized by means known in the art. The quantum dots described herein can be conjugated to any drug. The drug may or may not be therapeutically effective to any organism. In this regard, the quantum dots may be conjugated to a candidate drug wherein one of ordinary skill in the appropriate art reasonably believes that the candidate drug may have a therapeutic or beneficial effect to any whole organism. [0049] For example, biocompatible quantum dots can be prepared in which F₂ or F₅ represents a maleimide group. These maleimide-functionalized quantum dots can then bind to a thiol group of a biological agent, such as a protein. [0050] In another example, quantum dots with F₂ or F₅ surface groups can be biotinylated by using spacer molecules of the general structure: F₆-Ss-biotin, in which F₆ is a group that is reactive to F₂ or F₅, as described herein and S₅ is as described herein for Si_-4. Such biotinylated quantum dots can then be conjugated to suitable biological agents, such as biotinylated proteins (e.g., antibodies), via high affinity biotin-streptavidin or biotin-avidin linkages. [0051] In yet another example, crosslinking molecules comprising F₆-S₅-nitrilotriacetic acid (NTA) can to be added to quantum dots to provide reversible and specific binding to proteins. F₆ and S₅ are as described herein. After binding a suitable metal (e.g., Ni or Cu) to NTA, these chelator groups facilitate the binding to histidine (His) groups of polypeptides or His-tagged proteins. This binding reaction is reversible because the addition of another chelator (e.g., EDTA) results in the unbinding of the histidines from NTA. [0052] In yet another example, polypeptides of the form F₆-polypeptide are added to quantum dots comprising an inner inert layer and an outer inert layer. F₆ and the polypeptide are as described herein. Given the specific sequence of the peptide to be attached, peptide-nanoparticle conjugates can be employed to: (a) provide a convenient means of introducing an organic radiolabel (e.g., ³H or ¹⁴C) to the nanoparticle; (b) provide specific metal binding affinity for the sensing of transition metal ions in both biological and non-biological solutions; (c) provide the capability of generating bimodal ("dual labeled") imaging/radiopharmaceuticals that take advantage of the visual imaging properties of the nanoparticle and the ability of a particular peptide to bind radiopharmaceutically-active metals (e.g., ^99mTc, ²¹²Bi, ²¹³Bi, ²¹²Pb, ⁹⁰Y, ²²⁵Ac, ¹⁸⁶Re); and/or (d) allow the specific targeting of nanoparticles to subcellular locations and organelles via membrane-permeating TAT peptides attached to the quantum dot. Suitable procedures using radiopharmaceuticals can be found in the literature (see, for example, Mettler Jr. et al., Essentials of Nuclear Medicine Imaging, Grune and Stratton, Inc.: New York, 1983). [0053] The present inventive quantum dots are useful in a number of in vitro and in vivo methods, particularly, in the instance that the quantum dots are conjugated to a biological agent, such as a biomolecule or any drug. As used herein, the term "in vitro" means that the method does not take place within a host. As used herein, the term "in vivo" means that the method takes place within a host or any part thereof. These methods are further provided by the present invention. [0054] In this regard, the present invention provides a method of detecting a target in a sample. The method comprises (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, (ii) allowing the biological agent to specifically bind to the target, and (iii) analyzing the sample via spectroscopy, thereby obtaining a spectroscopic signature of the sample, wherein the spectroscopic signature is indicative of the presence or the absence of the target in the sample. [0055] The present invention also provides a method of detecting the location of a target within a sample. The method comprises (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the sample or a section thereof, thereby detecting the location of the target within the sample. [0056] Also provided by the present invention is a method of monitoring a biological process in vitro. The method comprises (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, wherein the target functions in a biological process, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the sample or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vitro. [0057] The present invention provides a method of detecting the location of a target in vivo. The method comprises (i) administering to a host a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the host, (ii) allowing the biological agent to specifically bind to the target, (iii) imaging the host, a section thereof, or a cell thereof, thereby detecting the location of the target in vivo. [0058] The present invention provides a method of monitoring a biological process in vivo. The method comprises (i) administering to a host a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the host, wherein the target functions in a biological process, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the host, a section, or a cell thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vivo. [0059] Further provided by the present invention is a- method of monitoring a biological process in vitro. The method comprises (i) contacting a sample with two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the sample, wherein each of the targets functions in a biological process, (ii) allowing the biological agents to specifically bind to the targets, and (iii) imaging the sample or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vitro. [0060] A method of detecting the location of more than one target in vivo is provided by the present invention. The method comprises (i) administering to a host two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the host, (ii) allowing the biological agents to specifically bind to the targets, (iii) imaging the host, a section thereof, or a cell thereof, thereby detecting the location of the more than one target in vivo. [0061] The present invention also provides a method of monitoring a biological process in vivo. The method comprises (i) administering to a host two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the host, wherein each of the targets functions in a biological process, (ii) allowing the biological agents to specifically bind to the targets, and (iii) imaging the host, a sample thereof, or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vivo. [0062] When two or more quantum dots are used for the methods of the present invention, preferably the two or more quantum dots are spectroscopically distinguishable. In other words, the emissive signals of the two or more quantum dots are sufficiently set apart from one another, such that meaningful analysis of the data can occur. In order to prepare two or more distinguishable quantum dots, the quantum dots, for example, can have different core sizes. Alternatively, the quantum dots can have different inner and/or outer inert layers, but the same core size. In preferred embodiments, the quantum dots have different core sizes. [0063] As used herein, the term "target" refers to any entity that specifically binds to a biological agent conjugated to a quantum dot. The target can be, for instance, a protein, a nucleic acid molecule, a fragment of either of the foregoing, a small-molecule drug, a cell, a tissue, or a drug metabolite. Suitable targets that are proteins include, but are not limited to, antibodies, or fragments thereof, peptides, hormones, growth factors, cytokines, tumor- associated proteins, cell-surface receptors, coagulation factors, proteins associated with a disease or a condition, and the like. One of ordinary skill in the art realizes that the phrase "specifically binds to" generally means that the binding occurs in such a manner that excludes the binding of most other entities within the sample or host. A target-biological agent binding interaction typically has a dissociation constant, KD, within the range of about micromolars to about picomolars. The phrase "allowing the biological agent to specifically bind to the target" as used herein refers to providing conditions under which the biological agent will specifically bind to the target. Such conditions are empirically determined by one of ordinary skill in the art by varying certain parameters, e.g., salt concentrations, pH, temperature, concentration of the target, concentration of the biological agent. One ordinarily skilled appreciates that these parameters affect the specific binding of the biological agent to the target. Typically, but not always, suitable conditions for allowing the biological agent to specifically bind to the target are physiological conditions, such that in the in vivo methods described herein, suitable conditions may be providing a sufficient period of time for the biological agent to specifically bind to the target. [0064] With respect to the present inventive in vitro methods, i.e., the method of detecting a target in a sample, the method of detecting more than one target in a sample, and the method of monitoring a biological process in vitro, the sample can be any sample, such as blood, lymph, ductal fluid, tissue, cell cultures, a single cell, urine, a biopsy, and the like. The sample can also be obtained from any source, such as a host, an animal, a cultured cell line, a plant, and a tumor. The terms "host" and "whole organism" as used herein refers to any living organism, including for example, bacteria, yeast, fungi, plants, and mammals. Preferably, the host is a mammal. For purposes of the present invention, mammals include, but are not limited to, the order Rodentia, such as mice, and the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human. [0065] In one embodiment of the invention, the source can represent a normal, undiseased state. Alternatively, the source, such as the mammal, has a disease or a condition, such that the method achieves detection or prognosis of the disease or the condition. In a preferred embodiment of the invention, the disease is cancer including, but not limited to, lung cancer, brain cancer, ovarian cancer, uterine cancer, testicular cancer, lymphoma, leukemia, stomach cancer, pancreatic cancer, skin cancer, breast cancer, adenocarcinoma, glioma, bone cancer, and the like. The present inventive methods of detecting cancer are particularly useful for detecting skin and breast tumors that are located close to the skin surface. [0066] In some of the present inventive in vitro methods described herein, the sample is analyzed via spectroscopy in order to obtain a spectroscopic signature. By "spectroscopy" as used herein is meant any technique for analyzing molecules based on how they absorb radiation. One of ordinary skill in the art realizes that many methods of spectroscopy are known in the art, including, for instance, ultraviolet-visible (UV-VIS) spectroscopy, infrared (IR) spectroscopy, fluorescence spectroscopy (e.g., single molecule fluorescence microscopy, fluorescence correlation spectroscopy, confocal microscopy), Raman spectroscopy, mass spectrometry, and nuclear magnetic resonance (NMR). For the present inventive methods, the sample preferably is analyzed via fluorescence spectroscopy. More preferably, the sample is analyzed via visible to infrared fluorescence spectroscopy and, most preferably, the sample is analyzed via far- red and near- infrared fluorescence. The term "spectroscopic signature" as used herein refers to a resulting pattern, plot, or spectrum obtained upon performing spectroscopy on a sample. The spectroscopic signature obtained of a sample containing a biological agent bound to a target can be compared to a control spectroscopic signature, wherein the target is not present in the sample or host. [0067] With respect to the present inventive methods of detecting a location of a target or detecting locations of more than one target, the term "location" as used herein refers to the physical position or site where the target is found within the sample or host. The location can be in reference to a cell, i.e., a sub-cellular location. Alternatively, the location of the target can be in reference to a tissue or an organ. The location of the target can also be in, reference to a whole organism, a whole plant or whole animal. The location can be on the surface of the host or animal or it can be within the host or animal. Preferably, the location of the target is deep within the animal or host, i.e., underneath several layers of tissue. [0068] The location of the target is determined via imaging the sample with the conjugated quantum dot bound to the target. Many methods of imaging are known in the art, including, for example, x-ray computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and optical imaging. Preferably, the imaging is done via fluorescence. More preferably, the imaging is done via visible to infrared fluorescence and, most preferably, the imaging is done through far-red and near-infrared fluorescence. One of ordinary skill in the art realizes that most, if not all, forms of imaging involve the detection of the wavelengths emitted by the quantum dot(s). Methods requiring imaging of the present inventive quantum dots can involve detection of near infrared or far red emission peak wavelengths. One ordinarily skilled also appreciates that this property of the quantum dots allow imaging of targets deep within a host or animal. [0069] The term "biological process" as used herein refers to any event, physiological or molecular, that occurs in or on the body of a host. The biological process can be, for instance, a molecular process (e.g., signal transduction pathway, a chemical reaction, an enzyme reaction, a binding reaction), a cellular process (e.g., mitosis, cytokinesis, cell motility, cell proliferation, cellular differentiation, cell lysis, endocytosis, phagocytosis, exocytosis, cell fusion), a physiological process (e.g., blood clot formation), and the like. The biological process can be one that occurs in response to a stimulus or the process can be one that occurs without stimulus and takes place over a period of time. A stimulus can be exogeneous (not naturally-occurring) or endogeneous (naturally-occurring) to the whole organism. The stimulus can vary in duration. It can be incessant or it can be a short event that occurs only once. It can also be a short, repeated stimulus. Suitable stimuli for use in the present inventive methods include, but are not limited to, an injection of a drug or a hormone, exposure to light, pain, electrical pulses, magnetic fields, temperature, and the like. [0070] The quantum dots described herein can be formed as a composition, such as a pharmaceutical composition. Pharmaceutical compositions containing the quantum dots can comprise more than one active ingredient, such as more than one quantum dot conjugated to a different biological agent. The pharmaceutical composition can alternatively comprise a quantum dot in combination with pharmaceutically active agents or drugs other than those conjugated to them. [0071] The compositions comprising the quantum dots can comprise a carrier, a diluent, or an excipient. The carrier can be any suitable carrier. Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical composition, the quantum dots of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. [0072] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. [0073] The choice of carrier will be determined in part by the particular quantum dot and biological agent conjugated thereto, as well as by the particular method used to administer the compound and/or inhibitor. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present inventive methods. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the quantum dots of the present invention are known, and, although more than one route can be used to administer a particular quantum dot, a particular route can provide a more immediate and more effective response than another route. [0074] Injectable formulations are among those formulations that are preferred in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). [0075] Topical formulations are well-known to those of skill in the art. Such formulations are particularly suitable in the context of the present invention for application to the skin. [0076] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the quantum dot dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art. [0077] The quantum dots, alone or in combination with each other and/or with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa. [0078] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The quantum dots can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [0079] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. [0080] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-b-aminopropionates, and 2- alkyl- imidazoline quaternary ammonium salts, and (e) mixtures thereof. [0081] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. [0082] Additionally, the quantum dots, or compositions comprising such compounds and/or inhibitors of Hsp90, can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. [0083] One of ordinary skill in the art will readily appreciate that the quantum dots of the present inventive methods can be modified in any number of ways, such that the efficacy of the quantum dot is increased through the modification. For instance, the quantum dot or the biological agent conjugated thereto could be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating quantum dots or biological agents to targeting moieties is known in the art. See, for instance, Wadwa et al., /. Drug Targeting 3: 111 (1995), and U.S. Patent No. 5,087,616. The term "targeting moiety" as used herein, refers to any molecule or agent that specifically recognizes and binds to a cell-surface receptor, such that the targeting moiety directs the delivery of the quantum dot and/or biological agent to a population of cells on which surface the receptor is expressed. Targeting moieties include, but are not limited to, antibodies, or fragments thereof, peptides, hormones, growth factors, cytokines, and any other naturally- or non- naturally-existing ligands, which bind to cell surface receptors. The term "linker" as used herein, refers to any agent or molecule that bridges the quantum dot or biological agent to the targeting moiety. One of ordinary skill in the art recognizes that sites on the quantum dot or biological agent, which are not necessary for the function of the quantum dot or biological agent, are ideal sites for attaching a linker and/or a targeting moiety, provided that the linker and/or targeting moiety, once attached to the quantum dot or biological agent, do(es) not interfere with the function of the quantum dot or biological agent, i.e., the ability to absorb and emit detectable energy or specifically bind to a target or targets. [0084] Alternatively, the quantum dots of the present invention can be modified into a depot form, such that the manner in which the quantum dot is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of quantum dots can be, for example, an implantable composition comprising the quantum dot and a porous material, such as a polymer, wherein the quantum dot is encapsulated by or diffused throughout the porous material. The depot is then implanted into the desired location within the body and the quantum dot is released from the implant at a predetermined rate by diffusing through the porous material. [0085] Furthermore, the present inventive methods can comprise the administration of the quantum dot(s), in the presence or absence of an agent that enhances its efficacy, or the methods can further comprise the administration of other suitable components, such as those that can protect the quantum dot and/or the biological agent from degradation within the host or those that can prevent the elimination from the host or cellular uptake of the quantum dot. [0086] For purposes of the present inventive methods, the amount or dose of the quantum dot(s) administered should be sufficient to effect a response in the animal over a reasonable time frame. Particularly, the dose of the quantum dot should be sufficient to allow the biological agent(s) to specifically bind to its target(s) within about 1-2 hours, if not 3-4 hours, from the time of administration. The dose will be determined by the efficacy of the particular quantum dot and/or biological agent conjugated thereto and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated. Many assays for determining an administered dose are known in the art. For purposes of the present invention, an assay, which comprises comparing the extent to which the biological agent(s) specifically bind(s) to its target(s) within the host upon administration of a given dose of a quantum dot to a mammal among a set of mammals that are each given a different dose of the quantum dot(s), could be used to determine a starting dose to be administered to a mammal. The extent to which the biological agent conjugated to the quantum dot specifically binds to the target within the host upon administration of a certain dose can be determined through imaging the host or a section thereof. [0087] The dose also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular quantum dot. Ultimately, the attending physician will decide the dosage of the compound or inhibitor of the present invention with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, quantum dot to be administered, and route of administration. [0088] Quantum dots prepared as described herein and that are conjugated to a biological agent can have multiple uses. For example, quantum dot-labeled antibodies can be prepared to use against a urokinase plasminogen activator receptor (uPAR) and α_vβ₃ integrin reconstituted into model biomembranes and cancer cells and study molecular properties using suitable optical imaging techniques (e.g., single molecule fluorescence microscopy, fluorescence correlation spectroscopy, confocal microscopy). Alternatively, surface-functionalized biocompatible quantum dots conjugated to metal binding peptides (e.g., a Cu-binding peptide) can be prepared for metal sensing applications. In another example, biocompatible quantum dots functionalized with a TAT-peptide, thereby ensuring membrane penetration, can be prepared to study interactions of TAT-peptides with TAR- RNA at the single molecule level. In yet another example, follicle-stimulating hormone (FSH) can be added to TAT-peptide-functionalized quantum dots to study molecular processes on ovarian cancer cells. In yet another example, biocompatible quantum dots carrying both the radio labels Te, which are bound to the quantum dot via Te-binding peptides, and particular antibodies for angiogenesis studies. [0089] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1 [0090] This example demonstrates the preparation of a QD with hydrophilic surface groups prepared by the "grafting to" process. [0091] Equimolar amounts of amino-tøs-ethyleneglycol (NH₂CH₂CH₂OCH₂CH₂OH) and l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) were stirred in aqueous MES buffer (pH 6) at 5 ⁰C for 5 minutes, after which time caboxylated quantum dots (approximately 10 mole %) were added and the solution was stirred for 5 more minutes. The derivatized dots were then precipitated with methanol, centrifuged, and washed several times with cold methanol. Approximately 85-95% recovery of dots was obtained. [0092] The resulting QD 's have a thin but highly compact PEG layer.

EXAMPLE 2 [0093] This example demonstrates the preparation of a QD with grafted PEG chains that are in a coil conformation.

[0094] Equimolar amounts of

and l-[3-(dimethylamino)propyl]- 3-ethylcarbodiimide hydrochloride (EDC) are stirred in aqueous MES buffer (pH 6) at 5 ⁰C, carboxylated quantum dots (approximately 10 mole %) are added and the solution is stirred. The derivatized dots are then precipitated, centrifuged, and washed several times.

EXAMPLE 3 [0095] This example demonstrates the preparation of a QD with an inner inert layer comprising two different moieties (Y-Si-Fi and Z-S₂-F₂). See Figure 8. [0096] Equimolar amounts of amino-b/s-ethyleneglycol (NH₂CH₂CH₂OCH₂CH₂OH), O H₂N-^CH ₂-O-^CH ₂-CH₂-C-O¹BU_{1 anc}i EDC are stirred in aqueous MES buffer (pH 6) at 5 ⁰C. O H₂N-^CH ₂-O-^CH ₂-CH₂-C — O'BU i_s deprotected with any suitable deprotecting agent (e.g., trifluoroacetic acid (TFA)). For example, suitable conditions include using 25 mol% TFA

in CH₂Cl₂ in an ice bath at 10 ⁰C. Upon deprotection

is added and the solution is stirred. Carboxylated quantum dots (approximately 10 mole %) are added and the solution is stirred again. The derivatized dots are then precipitated, centrifuged, and washed several times.

EXAMPLE 4 [0097] This example demonstrates the preparation of a QD with an inner inert layer using the "grafting from" approach. [0100] Equimolar amounts of amino-føs-ethyleneglycol o (NH₂CH₂CH₂OCH₂CH₂OH), H₂N-^CH ₂-O-^CH ₂-CH₂-C-O¹BU_{5 an}d EDC are stirred in aqueous o MES buffer (pH 6) at 5 ⁰C. H₂N-^CH ₂-₀-^CH ₂__CH₂-C— O'BU i_s deprotected with any suitable deprotecting agent (e.g., trifluoroacetic acid (TFA)). For example, suitable conditions include using 25 mol% TFA in CH₂Cl₂ in an ice bath at 10 °C. Upon deprotection a o mixture Of NH₂CH₂CH₂OCH₂CH₂OH and H₂N-^CH ₂-O-^CH ₂-CH₂-C-O¹BuOr O O H₂N-CH₂-O-CH₂-CH₂-C — O¹Bu alone is added. H₂N-CH₂-O-CH₂-CH₂-C — O¹Bu is deprotected again o and a final layer Of NH₂CH₂CH₂OCH₂CH₂OH and H₂ ^N-^CH ₂-O-^CH ₂-CH₂-C-O¹BuQr o H₂N-^CH ₂-O-^CH ₂-CH₂-C — O¹Bu alone is added.

EXAMPLE 5 [0101] This example demonstrates the preparation of a QD with an inner inert layer and an outer inert layer. [0102] Quantum dots with inner inert layers such as those prepared in Examples 1-4 are combined with F₄-S₄-F₅ (Figure 9), in which F₄ is reactive to Fi, F₂ and/or F₃, S₄ is a hydrophilic polymer, and F₅ is reactive to one or more biological agents.

[0103] AU references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0104] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention. [0105] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A quantum dot comprising (a) an inner inert layer; and (b) an outer inert layer, wherein the outer inert layer is optionally conjugated to a biological agent.

2. The quantum dot of claim 1, wherein the inner inert layer comprises X-Si-Fi, wherein X is reactive with a group Q found on the surface of the quantum, Si is a spacer, and Fi is a functional group that provides inert surface properties.

3. The quantum dot of claim 2, wherein X is amino, isothiocyano, haloacetyl, benzyl halide, alkyl halide, maleimido, aziridino, acryloyl, an acrylating agent, or a thiol- disulfide exchange reagent.

4. The quantum dot of claim 2, wherein Si is a hydrocarbon chain, a hydrophilic polymer, or a polypeptide.

5. The quantum dot of claim 1, wherein the outer inert layer comprises a hydrophilic polymer.

6. The quantum dot of claim 5, wherein the hydrophilic polymer is selected from the group consisting of hydroxyethyl cellulose, glycosaminoglycans, dextran, dextran sulfate, polyethyleneimine, polyacrylamide, polyester, polyvinyl alcohol (PVA), poly(N- vinylpyrrolidine) (PVP), poly(N-vinylamide), poly(N-vinyl saccharide), poly(aminoacrylate), poly(sodium acrylate), poly(sodium methacrylate), poly(sodium styrenesulfonate,) polyurethane, polyethylene glycol (PEG), polyethylene oxide (PEO), poly(2-hydroxyethyl methacrylate) (poly HEMA), poly(2-(hydroxyethoxy)ethyl methacrylate) (poly DEGMA), poly(2-(acetoxy)ethyl methacrylate (poly AEMA), mixtures thereof, and copolymers thereof.

7. The quantum dot of claim 1, wherein the quantum dot is conjugated to a biological agent.

8. The quantum dot of any of claims 1-6, wherein the biological agent is a biomolecule.

9. The quantum dot of claim 8, wherein the biomolecule is selected from the group consisting of a protein, a peptide, a nucleic acid molecule, and a combination thereof.

10. A composition comprising the quantum dot of claim 1 and a carrier.

11. A method of detecting a target in a sample comprising (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, (ii) allowing the biological agent to specifically bind to the target, and (iii) analyzing the sample via spectroscopy, thereby obtaining a spectroscopic signature of the sample, wherein the spectroscopic signature is indicative of the presence or the absence of the target in the sample.

12. A method of detecting the location of a target within a sample comprising (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the sample or a section thereof, thereby detecting the location of the target within the sample.

13. A method of monitoring a biological process in vitro comprising (i) contacting a sample with a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the sample, wherein the target functions in a biological process, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the sample or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vitro.

14. A method of detecting the location of a target in vivo comprising (i) administering to a host a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the host, (ii) allowing the biological agent to specifically bind to the target, (iii) imaging the host, a section thereof, or a cell thereof, thereby detecting the location of the target in vivo.

15. A method of monitoring a biological process in vivo comprising (i) administering to a host a quantum dot comprising an inner inert layer and an outer inert layer, which is conjugated to a biological agent, wherein the biological agent specifically binds to a target in the host, wherein the target functions in a biological process, (ii) allowing the biological agent to specifically bind to the target, and (iii) imaging the host, a section, or a cell thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vivo.

16. A method of monitoring a biological process in vitro comprising (i) contacting a sample with two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the sample, wherein each of the targets functions in a biological process, (ii) allowing the biological agents to specifically bind to the targets, and (iii) imaging the sample or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vitro. 0/3 26

17. A method of detecting the location of more than one target in vivo comprising (i) administering to a host two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the host, (ii) allowing the biological agents to specifically bind to the targets, (iii) imaging the host, a section thereof, or a cell thereof, thereby detecting the location of the more than one target in vivo.

18. A method of monitoring a biological process in vivo comprising (i) administering to a host two or more quantum dots each comprising an inner inert layer and an outer inert layer, wherein each of the quantum dots is conjugated to a different biological agent, wherein each of the biological agents specifically binds to a different target in the host, wherein each of the targets functions in a biological process, (ii) allowing the biological agents to specifically bind to the targets, and (iii) imaging the host, a sample thereof, or a section thereof over a period of time or before and after a stimulus, thereby monitoring a biological process in vivo.